LOCUS       sequence-306145-        3906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SENP_Eu protease test substrate 
            H14-SUMO_Eu1-MBP-3xFlagTag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3906)
  AUTHORS   Vera Rodriguez A, Frey S, Gorlich D
  TITLE     Engineered SUMO/protease system identifies Pdr6 as a bidirectional 
            nuclear transport receptor.
  JOURNAL   J Cell Biol. 2019 Jun 3;218(6):2006-2020. doi: 
            10.1083/jcb.201812091. Epub 2019 Apr 25.
  PUBMED    31023724
REFERENCE   2  (bases 1 to 3906)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1083/jcb.201812091"; journalName: "J Cell Biol"; date: 
            "2019-06-3- 3"; volume: "218"; issue: "6"; pages: "2006-2020"
COMMENT     Sequence Label: pAV0279
FEATURES             Location/Qualifiers
     source          1..3906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     protein_bind    62..78
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             499..1596
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1453..1462
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1570..1593
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1612..1677
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     terminator      1697..1783
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1818..1835)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1952..2497)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2032..2051)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2611..2705)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2749..3540)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     primer_bind     3874..3892
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aaccatgagc
      121 aagcatcacc atcattcagg ccatcaccat accggacacc accatcattc aggcagtcat
      181 caccattccg gatctgctgc gggtggcgaa gaagataaga aaccggcagg tggcgaaggt
      241 ggcggtgccc atatcaacct gaaagtgaaa ggtcaagacg gcaacgaagt ctttttccgc
      301 atcaaacgtt ctacccagct gaaaaagctg atgaacgcat actgtgaccg tcagtctgta
      361 gacatgaagg caattgcttt cctctttaag ggtcgtcgcc tacgtgcgga aaggaccccg
      421 gatgaactgg aaatggaaga tggcgacgaa atcgacgcaa tgctgcacca gactggaggc
      481 gccggtaccg gtactagtaa aactgaagaa ggtaaactgg taatctggat taacggcgat
      541 aaaggctata acggtctcgc tgaagtcggt aagaaattcg agaaagatac cggaattaaa
      601 gtcaccgttg agcatccgga taaactggaa gagaaattcc cacaggttgc ggcaactggc
      661 gatggccctg acattatctt ctgggcacac gaccgctttg gtggctacgc tcaatctggc
      721 ctgttggctg aaatcacccc ggacaaagcg ttccaggaca agctgtatcc gtttacctgg
      781 gatgccgtac gttacaacgg caagctgatt gcttacccga tcgctgttga agcgttatcg
      841 ctgatttata acaaagatct gctgccgaac ccgccaaaaa cctgggaaga gatcccggcg
      901 ctggataaag aactgaaagc gaaaggtaag agcgcgctga tgttcaacct gcaagaaccg
      961 tacttcacct ggccgctgat tgctgctgac gggggttatg cgttcaagta tgaaaacggc
     1021 aagtacgaca ttaaagacgt gggcgtggat aacgctggcg cgaaagcggg tctgaccttc
     1081 ctggttgacc tgattaaaaa caaacacatg aatgcagaca ccgattactc catcgcagaa
     1141 gctgccttta ataaaggcga aacagcgatg accatcaacg gcccgtgggc atggtccaac
     1201 atcgacacca gcaaagtgaa ttatggtgta acggtactgc cgaccttcaa gggtcaacca
     1261 tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg ccgccagtcc gaacaaagag
     1321 ctggcaaaag agttcctcga aaactatctg ctgactgatg aaggtctgga agcggttaat
     1381 aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg aggaagagtt ggcgaaagat
     1441 ccacgtattg ccgccaccat ggaaaacgcc cagaaaggtg aaatcatgcc gaacatcccg
     1501 cagatgtccg ctttctggta tgccgtgcgt actgcggtga tcaacgccgc cagcggtcgt
     1561 cagactgtcg atgaagccct gaaagacgcg cagactaatg gatctggttc tgattataaa
     1621 gatcatgatg gcgattataa agatcatgat attgattata aagatgatga tgataaataa
     1681 gcttctctag aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     1741 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgc cctagaccta
     1801 gggatatatt ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga
     1861 gcggaaatgg cttacgaacg gggcggagat ttcctggaag atgccaggaa gatacttaac
     1921 agggaagtga gagggccgcg gcaaagccgt ttttccatag gctccgcccc cctgacaagc
     1981 atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc
     2041 aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg
     2101 gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg
     2161 taggcagttc gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc
     2221 gccttatccg gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg
     2281 gcagcagcca ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg
     2341 ctaaactgaa aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa
     2401 agagttggta gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc
     2461 agagcaagag attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat
     2521 aaaatatttc tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc
     2581 catacgatat aagttgttgc tagtgcttgg attctcacca ataaaaaacg cccggcggca
     2641 accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag
     2701 gagtccaagc gagctcgata tccgtcggct tgaacgaatt gttagacatt atttgccgac
     2761 taccttggtg atctcgcctt tcacgtagtg gacaaattct tccaactgat ctgcgcgcga
     2821 ggccaagcga tcttcttctt gtccaagata agcctgtcta gcttcaagta tgacgggctg
     2881 atactgggcc ggcaggcgct ccattgccca gtcggcagcg acatccttcg gcgcgatttt
     2941 gccggttact gcgctgtacc aaatgcggga caacgtaagc actacatttc gctcatcgcc
     3001 agcccagtcg ggcggcgagt tccatagcgt taaggtttca tttagcgcct caaatagatc
     3061 ctgttcagga accggatcaa agagttcctc cgccgctgga cctaccaagg caacgctatg
     3121 ttctcttgct tttgtcagca agatagccag atcaatgtcg atcgtggctg gctcgaagat
     3181 acctgcaaga atgtcattgc gctgccattc tccaaattgc agttcgcgct tagctggata
     3241 acgccacgga atgatgtcgt cgtgcacaac aatggtgact tctacagcgc ggagaatctc
     3301 gctctctcca ggggaagccg aagtttccaa aaggtcgttg atcaaagctc gccgcgttgt
     3361 ttcatcaagc cttacggtca ccgtaaccag caaatcaata tcactgtgtg gcttcaggcc
     3421 gccatccact gcggagccgt acaaatgtac ggccagcaac gtcggttcga gatggcgctc
     3481 gatgacgcca actacctctg atagttgagt cgatacttcg gcgatcaccg cttccctcat
     3541 gatgtttaac tttgttttag ggcgactgcc ctgctgcgta acatcgttgc tgctccataa
     3601 catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg gatgcccgag gcatagactg
     3661 taccccaaaa aaacagtcat aacaagccat gaaaaccgcc actgcgccgt taccaccgct
     3721 gcgttcggtc aaggttctgg accagttgcg tgagcgcata cgctacttgc attacagctt
     3781 acgaaccgaa caggcttatg tccactgggt tcgtgccttc atccggatat cgacgtctaa
     3841 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     3901 cttcac
//
